Carrier Molecule Compositions and Related Methods

ABSTRACT

A carrier molecule composition. Specific implementations may include: a carrier molecule including at least one cell penetrating peptide (CPP) where the carrier molecule may include at least one hydrophobic domain and where the carrier is non-covalently associated with a biologically active molecule in one of a micelle and a liposome.

CROSS REFERENCE TO RELATED APPLICATIONS

This document claims the benefit of the filing date of U.S. ProvisionalPatent Application 62/088,813 entitled “Carrier Molecule Compositionsand Related Methods” to Tan et al. which was filed on Dec. 8, 2014, (the'813 provisional) the disclosure of which is hereby incorporatedentirely herein by reference.

SEQUENCE LISTING

This document contains the material in and hereby incorporates entirelyherein by reference the sequence listing file in ASCII text format filedon Dec. 8, 2015 named SequenceListing003_ST25.txt, created Dec. 8, 2015,which is 11,515 bytes in size.

BACKGROUND

1. Technical Field

Aspects of this document relate generally to compositions and methodsused to deliver biologically active molecules. More specificimplementations involve include compositions that are capable ofdelivering biologically active molecules transdermally.

2. Background

Biologically active molecules can carry out, participate in, or initiatevarious biochemical changes in a human or animal body. For example,botulinum toxin is a biologically active molecule that acts as aneurotoxin that can cause muscle paralysis and is used to treat wrinklesof the skin. Botulinum toxin is conventionally administered to the skinthrough injection. Additional background information regarding thestructure of skin, wrinkles of the skin, treatment methods for wrinkles,and botulinum toxin may be found in the following references, thedisclosures of each of which are hereby incorporated entirely herein byreference: Inlander, Skin, New York, N.Y.: People's Medical Society, p.1-7 (1998); Benedetto, International Journal of Dermatology, V. 38, p.641-655 (1999); Stegman et al., “The Skin of the Aging Face,” CosmeticDermatological Surgery, 2nd ed., St. Louis, Mo.: Mosby Year Book: p.5-15 (1990); Lamanna, Science, V. 130, p. 763-772 (1959); Baron et al.,Bailey & Scotts Diagnostic Microbiology, St. Louis, Mo.: Mosby YearBook, p. 504-523 (1994); Carruthers and Carruthers, Adv. Dermatol., V.12, p. 325-348 (1997); Markowitz, Hunters Tropical Medicine, 7^(th) Ed.,Philadelphia: W. B. Saunders, p. 441-444 (1991); Schantz and Scott,Biomedical Aspects of Botulinum, New York: Academic Press, p. 143-150(1981); and Scott, Ophthalmol, V. 87, p. 1044-1049 (1980).

SUMMARY

Implementations of compositions may include: a carrier moleculeincluding at least one cell penetrating peptide (CPP) where the carriermolecule may include at least one hydrophobic domain where the carrieris non-covalently associated with a biologically active molecule in oneof a micelle and a liposome.

Implementations of compositions may include one, all, or any of thefollowing:

The carrier molecule may be amphiphilic.

The carrier molecule may include at least one carbohydrate moiety.

The carrier molecule may include at least one alkyl chain.

The carrier molecule may include at least three hydrophobic amino acids.

The carrier molecule may include at least one phenylalanine.

The carrier molecule may include palmitoyl-gly_(p)-KKRPKPG (SEQ ID NO:5), octanoyl-gly_(p)-KKRPKPG (SEQ ID NO: 6), oleyl-gly_(p)-KKRPKPG (SEQID NO: 7) or any combination thereof, where p is an integer from 0 to20.

The carrier molecule may be selected from the group consisting ofFFFILVF-gly_(p)-KKRPKPG (SEQ ID NO: 1), FLVFFF-gly_(p)-KKRPKPG (SEQ IDNO: 2), KKRPKPG-gly_(p)-FLVFFF (SEQ ID NO: 3), or any combinationthereof, where p is an integer from 0 to 10.

The at least one CPP may be selected from the group consisting of anHIV-TAT fragment selected from the group consisting of a fragment thathas the formula (gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 8), afragment that has the formula (gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ IDNO: 9), a fragment that has the formula (gly)_(p)-RKKRRQRRR-(gly)_(q)(SEQ ID NO: 10), wherein the subscripts p and q are each independentlyan integer from 0 to 20; KKRPKPG (SEQ ID NO: 17); AAVLLPVLLAAP (SEQ IDNO: 15), or any combination thereof.

The biologically active molecule may be selected from the groupconsisting of VGVAPG (SEQ ID NO: 26); palmitoyl-TTS; retinyl retinoate;retinoic acid; steroid and steroidal compounds; hydroquinone; hyalonuricacid; non-steroidal anti-inflammatory drugs (NSAIDs) including naproxen,ibuprofen, and acetaminophen; skin-tightening peptides; lightactivatable moieties and compounds; ultraviolet (UV) light absorbing,blocking, or reflecting compounds; vitamins; cholesterol; drugs whichblock, influence, or interfere with neurotransmitter (such asacetylcholine) function; or any combination thereof.

The biologically active molecule may be selected from the groupconsisting of a botulinum toxin serotype selected from the groupconsisting of A, B, C, D, E, F, and G; recombinant botulinum toxin; amodified botulinum toxin; a fragment of a botulinum toxin; or anycombination thereof.

Implementations of a composition may include a biologically activemolecule and a carrier molecule including at least one lipophilic domainand at least one CPP where the carrier molecule further includes atleast one carbohydrate moiety, at least one alkyl chain, at least threehydrophobic amino acids, or any combination thereof. The carriermolecule and biologically active molecule may associated non-covalently.

Implementations of a composition may include one, all, or any of thefollowing:

The carrier molecule may include palmitoyl-gly_(p)-KKRPKPG (SEQ ID NO:5), octanoyl-gly_(p)-KKRPKPG (SEQ ID NO: 6), oleyl-gly_(p)-KKRPKPG (SEQID NO: 7) or any combination thereof, where p is an integer from 0 to20.

The carrier molecule may be selected from the group consisting ofFFFILVF-gly_(p)-KKRPKPG (SEQ ID NO: 1), FLVFFF-gly_(p)-KKRPKPG (SEQ IDNO: 2), KKRPKPG-gly_(p)-FLVFFF (SEQ ID NO: 3), or any combinationthereof, where p is an integer from 0 to 10.

The at least one CPP may be selected from the group consisting of anHIV-TAT fragment selected from the group consisting of a fragment thathas the formula (gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 8), afragment that has the formula (gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ IDNO: 9), a fragment that has the formula (gly)_(p)-RKKRRQRRR-(gly)_(q)(SEQ ID NO: 10), wherein the subscripts p and q are each independentlyan integer from 0 to 20; KKRPKPG (SEQ ID NO: 17); AAVLLPVLLAAP (SEQ IDNO: 15), or any combination thereof.

The biologically active molecule may be selected from the groupconsisting of VGVAPG (SEQ ID NO: 26); palmitoyl-TTS; retinyl retinoate;retinoic acid; steroid and steroidal compounds; hydroquinone; hyalonuricacid; non-steroidal anti-inflammatory drugs (NSAIDs) including naproxen,ibuprofen, and acetaminophen; skin-tightening peptides; lightactivatable moieties and compounds; ultraviolet (UV) light absorbing,blocking, or reflecting compounds; vitamins; cholesterol; drugs whichblock, influence, or interfere with neurotransmitter (such asacetylcholine) function; or any combination thereof.

The biologically active molecule may be selected from the groupconsisting of a botulinum toxin serotype selected from the groupconsisting of A, B, C, D, E, F, and G; recombinant botulinum toxin; amodified botulinum toxin; a fragment of a botulinum toxin; or anycombination thereof.

A kit for administration of botulinum toxin to a patient may include abotulinum toxin, an effective amount for transdermal delivery thereof ofa carrier molecule including at least one CPP where the carrier moleculeincludes at least one hydrophobic domain. A pH buffer system adapted tomaintain pH between 4.0 to 8.3 may be included along with a deviceincluding the botulinum toxin, the carrier molecule, and the pH buffersystem. The device may be adapted to administer the botulinum toxin to apatient via the patient's skin. The carrier molecule may benon-covalently associated with the botulinum toxin in one a micelle anda liposome. The device, the carrier molecule, the pH buffer system, orany combination thereof may be adapted to provide a controlled releaseof the botulinum toxin.

Implementations of a kit may include one, all, or any of the following:

The device may be a skin patch.

The botulinum toxin, the carrier molecule, and the pH buffer system maybe included in a liquid, gel, cream, lotion, and ointment coupled withthe device.

The foregoing and other aspects, features, and advantages will beapparent to those artisans of ordinary skill in the art from theDESCRIPTION and DRAWINGS, and from the CLAIMS.

DESCRIPTION

This disclosure, its aspects and implementations, are not limited to thespecific components, assembly procedures or method elements disclosedherein. Many additional components, assembly procedures and/or methodelements known in the art consistent with the intended carrier moleculecompositions, kits, and related method implementations will becomeapparent for use with particular implementations from this disclosure.Accordingly, for example, although particular implementations aredisclosed, such implementations and implementing components may compriseany shape, size, style, type, model, version, measurement,concentration, material, quantity, method element, step, and/or the likeas is known in the art for such carrier molecule compositions, kits, andrelated methods, and implementing components and methods, consistentwith the intended operation and methods.

The skin is the largest organ of the body and includes various layers,including the epidermis and dermis. The epidermis consists ofkeratinocytes and is divided into several layers based on their state ofdifferentiation. The epidermis can be further classified into thestratum corneum and the viable epidermis, which consists of the granularmelphigian and basal cells. The stratum corneum is hygroscopic andrequires at least 10% moisture by weight to maintain its flexibility andsoftness. The hygroscopicity is attributable in part to thewater-holding capacity of keratin. When the horny layer loses itssoftness and flexibility it becomes rough and brittle, resulting in dryskin. The dermis the thickest of the three layers of the skin andcontains most of the skin's structures, including various sweat and oilglands, hair follicles, nerve endings, and blood and lymph vessels. Themajor components of the dermis are collagen and elastin.

The pH of skin is normally between 5 and 6 due to the presence ofamphoteric amino acids, lactic acid, and fatty acids from the secretionsof the sebaceous glands. The term “acid mantle” refers to the presenceof the water-soluble substances on most regions of the skin. Thebuffering capacity of the skin is due in part to these secretions storedin the skin's horny layer.

One of the principal functions of skin is to provide a barrier to thetransportation of water and substances potentially harmful to normalhomeostasis. The body would rapidly dehydrate without a tough,semi-permeable skin. The skin helps to prevent the entry of harmfulsubstances into the body. Although most substances cannot penetrate thebarrier, a number of strategies have been developed to selectivelyincrease the permeability of skin with variable success.

As individuals age, their skin begins to wrinkle as the result of theoperation of various factors including biochemical, histological, andphysiologic changes that accumulate from environmental damage, theconstant pull of gravity, and repeated facial movements caused bycontraction of facial muscles.

Botulinum toxins (also known as botulin toxins or botulinum neurotoxins)are neurotoxins produced by the gram-positive bacteria Clostridiumbotulinum. Without being bound by any theory, they are believed act toproduce flaccid paralysis of muscles by preventing synaptic transmissionor release of acetylcholine across the neuromuscular junction, as wellas via other mechanisms.

Botulinum toxin is classified into eight neurotoxins that are distinctbut serologically related. Of these, seven can cause paralysis:botulinum neurotoxin serotypes A, B, C, D, E, F and G. The molecularweight of the botulinum toxin protein molecule for all seven of theseactive botulinum toxin serotypes is about 150 kilo Daltons (kD). Asreleased by the bacterium, the botulinum toxins are complexes comprisingthe 150 kD botulinum toxin protein molecule in question along withassociated non-toxin proteins. The complexes which have a molecularweight greater than about 150 kD are believed to contain a non-toxinhemaglutinin protein and a non-toxin and nonhemaglutinin protein. Thesetwo non-toxin proteins (which along with the botulinum toxin moleculecomprise the relevant neurotoxin complex) may act to provide stabilityagainst denaturation to the botulinum toxin molecule and protectionagainst digestive acids when toxin is ingested.

Because botulinum toxin is capable of producing muscle paralysis andinterfering with synaptic transmissions, it can be effectively used totreat various cosmetic skin conditions such as wrinkles as well as otherphysical and neurological conditions. As used herein, a biologicallyactive molecule is a molecule that, by non-limiting example, similarlyto botulinum toxin, chemically interacts with the biological tissueand/or cells to produce a desired biological effect. Botulinum toxin isan example of a biologically active molecule. Other examples ofbiologically active molecules include retinoic acid, steroids,hydroquinone, hyaluronic acid, and non-steroidal anti-inflammatory drugs(NSAIDs) including acetaminophen, ibuprofen, and naproxen.

Implementations of compositions of carrier molecules that act totransport and/or facilitate transport of biologically active moleculesinto cells and other tissues are disclosed herein. Also disclosed areimplementations of various kits containing various carrier moleculecompositions and implementations of various methods of administeringbiologically active molecules using carrier molecule compositions.

Implementations of carrier molecules disclosed herein contain at leastone lipophilic domain and at least one cell penetrating peptide. Invarious implementations, the lipophilic domain is a hydrophobic domain.The carrier molecule implementations are also designed so that thecarrier molecule directly and/or non-covalently associates with one ormore biologically active molecules. In particular implementations,carrier molecules associate noncovalently with one or more hydrophobic,lipophilic, or amphiphilic portions of another molecule. Such a moleculecan be the biologically active molecule(s) or a part of a deliverysystem for the carrier molecules which contains the biologically activemolecule(s).

In particular implementations, the carrier molecule compositions may beused on conjunction with a biologically active molecule which is abotulinum toxin. Such implementations may include those that enable thetransport or delivery of a botulinum toxin through the skin orepithelium without the use of transcutaneous delivery via a needle orinjection. These composition implementations may be used as topicalapplications for providing a botulinum toxin to a subject for varioustherapeutic, aesthetic and/or cosmetic purposes, as described herein.

In such implementations, the composition includes a botulinum toxinassociated with a carrier molecule where carrier molecule has at leastone lipophilic domain and/or hydrophobic domain and at least one cellpenetrating protein/peptide (CPP). In these implementations, theassociation between the carrier molecule and the botulinum toxin isnon-covalent. In various other implementations, the association betweenthe carrier molecule and the biologically active molecule may benon-covalent as well. In particular implementations, the lipophilicdomain may not be a polycation or polyanion. In various implementations,the lipophilic domain does not exert biologic activity itself but merelyfacilitates the transport of the biologically active molecule.

In various implementations, a wide variety of various molecules may beused as carrier molecules. The carrier molecules may have any of a widevariety of properties including being lipophilic or amphiphilic. Inparticular implementations, the carrier molecules may include acarbohydrate moiety. Examples of carbohydrate moieties that may beincluded in various implementations are, by non-limiting example,heptanoic acid, octanoic acid, palmitic acid, oleic acid, and any otherfatty acid, fatty alcohol, or other carbohydrate compound,Implementations may also include at least one alkyl chain. In suchimplementations, the alkyl chain may include at least 5 carbons, havebetween 50 and 30 carbons, or have between 6 and 14 carbons. The alkylchain may be fully or partially saturated. In implementations where thechain is only partially saturated, the carrier molecule implementationsmay include a mixture of cis/trans isomerized molecules or only the cisor only the trans isomer of the molecule.

Other carrier molecule implementations may include various combinationsof amino acids. In some implementations, at least three hydrophobicamino acids may be employed. In such implementations, at least one ofthe amino acids may be phenylalanine. In other implementations, thecarrier molecule may include at least 5 amino acids or at least 7 aminoacids selected from the group consisting of valine (V), phenylalanine(F), alanine (A), glycine (G), proline (P), methionine (M), tryptophan(W), leucine (L), and isoleucine (I), and combination thereof, and anyother hydrophobic amino acid. In other implementations, the carriermolecule may include at least 5 amino acids or at least 7 amino acidsselected from the group consisting of cysteine (C), serine (S), tyrosine(Y), glutamine (Q), threonine (T), asparagine (N), glutamate (E), lysine(K), aspartic acid (D), arginine (R), and histidine (H), any combinationthereof, and any other hydrophilic or polar amino acid. In someimplementations, combinations of hydrophobic and hydrophobic,hydrophilic, and/or polar amino acids may be used. Also, othernon-standard amino acids other than those listed may be utilized invarious implementations.

In implementations of carrier molecules that include just amino acids,various combinations may be used. In various implementations, thecarrier molecule may include a backbone selected from a peptidyllipophilic polymeric backbone, peptidyl lipophilic oligomeric backbone,a nonpeptidyl lipophilic polymeric backbone, and a nonpeptidyllipophilic oligomeric backbone.

As an example, the carrier molecule may be selected fromFFFILVF-gly_(p)-KKRPKPG (SEQ ID NO: 1), FLVFFF-gly_(p)-KKRPKPG (SEQ IDNO: 2), KKRPKPG-gly_(p)-FLVFFF (SEQ ID NO: 3), KKRPKPG (SEQ ID NO: 4),or any combination thereof, where p is an integer from 0 to 10. Sequencelistings corresponding with these carrier molecule implementations alongwith sequence listings as indicated throughout this document may be inthe Sequence Listing filed herewith, the disclosure of which is herebyincorporated entirely herein by reference. In various other carriermolecule implementations, various combinations of amino acids andpalmitoyl, octanoyl, and oleyl groups may be utilized. For example, thecarrier molecule may be one of palmitoyl-gly_(p)-KKRPKPG (SEQ ID NO: 5),octanoyl-gly_(p)-KKRPKPG (SEQ ID NO: 6), oleyl-gly_(p)-KKRPKPG (SEQ IDNO: 7), or any combination thereof, where p is an integer from 0 to 20.

In particular implementations, the carrier molecule includes alipophilic oligo- or polymeric backbone comprising at least onecovalently bonded CPP. Any of a wide variety of CPPs may be employed invarious composition implementations and in combination with variouscarrier molecule and active molecule implementations disclosed herein.One or more CPPs may be used, and may be included on the carriermolecule, active molecule, or both the carrier molecule and activemolecule.

For example, the CPP may be an HIV fragment or HIV-TAT or HIV-TATfragment. Example of such fragments include(gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 8), a fragment that has theformula (gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 9), a fragment thathas the formula (gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 10), and anycombination of such fragments, where the subscripts p and q are eachindependently an integer from 0 to 20. In particular implementations,the HIV-TAT fragment may be specifically the SEQ ID NO: 10 version. Inother implementations, the R9 CPP may be used, coded as RRRRRRRRR (SEQID NO: 11).

Other CPPs that may be included are Antennapedia, coded asRQIKWFQNRRMKWKK (SEQ ID NO: 12); Transduction Domain 1 (TD1) coded asNPGGYCLTKWMILAAELKCFGNTAVAKCNVNHDAEFCD (SEQ ID NO: 13); melittin, codedas GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 14); and prion, coded asAAVLLPVLLAAP (SEQ ID NO: 15). Other CPPs that may be employed include(gly)_(p)-KKRPKPG-(gly)_(q) (SEQ ID NO: 16) wherein the subscripts p andq are each independently an integer from 0 to 20; and KKRPKPG (SEQ IDNO: 17) standing alone. In particular implementations, the CPP may beFLVFFFGG (SEQ ID NO: 18). In other implementations the CPP may be-(Gly)_(n1)-(Arg)_(n2) (SEQ ID NO: 19) or gly_(n1a)-KKRPQPD-gly_(n1b)(SEQ ID NO: 20) where the subscript n1 is an integer of from 0 to about20, subscripts n1a and n2a are each integers of from 0 to about 20, andthe subscript n2 is an odd integer of from about 5 to about 25.

A wide variety of other CPPs from viral sources, including anyhomologous sequence from any virus capable of penetrating a human oranimal cell wall, may be included and/or employed in variousimplementations. These may be any currently known or hereafterdiscovered consistent with the principles disclosed herein.

In addition, a wide variety of synthetic or otherwise man-made CPPs maybe employed in various implementations. These may include one, acombination of all, or any combination of the following:KKRPKPGGGGFFFILVF (SEQ ID NO: 21), FFFILVFGGGKKRPKPG (SEQ ID NO: 22),GGGGKKRPKPG (SEQ ID NO: 23), RKKRRQRRRGGGGFFFILVF (SEQ ID NO: 24), andGGGGRKKRRQRRR (SEQ ID NO: 25). In particular implementations,GGGGKKRPKPG and GGGGRKKRRQRRR may be bonded to a palmitoyl group to formthe complete structure of the CPP and/or carrier molecule+CPP. Theparticular palmitoyl group used in various implementations of carriermolecules and CPPs disclosed herein may be n-palmitoyl.

A wide variety of biologically active molecules may be used in variousimplementations of compositions disclosed herein other than botulinumtoxin actives. These include, by non-limiting example, VGVAPG (SEQ IDNO: 26); palmitoyl covalently bonded to the TAT sequence disclosedherein as SEQ. NO. 10; retinyl retinoate; retinoic acid; steroid andsteroidal compounds; hydroquinone; hyalonuric acid; non-steroidalanti-inflammatory drugs (NSAIDs) including naproxen, ibuprofen, andacetaminophen; skin-tightening peptides; light activatable moieties andcompounds; ultraviolet (UV) light absorbing, blocking, or reflectingcompounds; vitamins; cholesterol; drugs which block, influence, orinterfere with neurotransmitter (such as acetylcholine) function; andany combination thereof.

A wide variety of composition implementations containing carriermolecules, CPPs, and biologically active molecules may be constructedusing the principles disclosed herein. As an initial step, the carriermolecules are first prepared. Each carrier molecule may include one ormore CPPs covalently bonded to it or otherwise associated with it.Following preparation of the carrier molecule/CPP combined molecule, thebiologically active molecules are then associated non-covalently withthe carrier molecule/CPP combined molecule. This may be done in a widevariety of ways, including, by non-limiting example, simple mixing,titrating, chelating, protein folding, incorporating into a liposome,incorporating into a nanoemulsion, direct associating, emulsifying, andany other technique for non-covalently associating the biologicallyactive molecule with the carrier molecule/CPP combined molecule.

Particular implementations may be prepared for delivery via an emulsionor a liposomal preparation. Emulsion preparations involve those carriermolecules and/or CPPs that contain predominately hydrophilic or polaramino acids and involve adhering/associating the carrier moleculesand/or CPPs (and correspondingly, the non-covalently bonded biologicallyactive molecules) to a plurality of micelles in the emulsion. Theemulsion may then be mixed with additional components contained in oneor more liquid/solid phases to form a final composition adapted to beapplied to a patient's skin.

Liposomal preparations may be used for those carrier molecules and/orCPPs that contain predominately hydrophobic amino acids. The combinationof the carrier molecule, one or more CPPs, and one or more biologicallyactive molecules is packaged into a liposome. The liposomes used invarious implementations may be those from, and/or manufactured accordingto the processes and technologies used by Encapsula NanoSciences ofBrentwood, Tenn.; Lippomix, Inc. of Novato, Calif.; Azaya TherapeuticsIncorporated of San Antonio, Tex.; Oakwood Laboratories, L.L.C. ofOakwood Village, Ohio; Tergus Pharma of Durham, N.C. The respectivedisclosures of such liposomal compositions, and manufacture processesand technologies for each manufacturer are included in Appendices A, B,C, D, and E, to the '813 provisional, the disclosure of which waspreviously hereby incorporated entirely herein by reference. A pluralityof liposomes are then prepared and mixed with additional components inone or more liquid/solid phases to form a final composition that alsoconfigured to be applied to patient's skin.

The final form of the composition implementations disclosed herein maytake the form of a liquid, gel, cream, lotion, or ointment. Thecomposition implementations may be stable when under room temperaturestorage and/or refrigerated storage. The compositions may have a pH fromabout 4.0 to about 8.3. In particular implementations, the compositionmay include a pH buffer system, which may include various components,including, by non-limiting example, ascorbate, citrate, phosphate, anycombination thereof, and any other pH buffering composition or compound.Particular composition implementations may be designed to provide acontrolled and/or time delayed release of the biologically activemolecule. This may be done in a variety of ways, including, bynon-limiting example, causing a concentration-dependent reaction betweenthe carrier molecule and/or CPP and a component of the skin to releasethe biologically active molecule, using a dissociation reaction with aparticular activation energy between the biologically active moleculeand the carrier molecule to drive a specific dissociation rate for thebiologically active molecules, utilizing a combination of CPPs attachedto various carrier molecules that have different reaction rates withskin cells to release biologically active molecules over time, and anyother chemical reaction-driven, mass transport, or energy transportdriven process designed to gradually release the biologically activemolecules to the desired tissues in the skin.

Implementations of compositions like those disclosed herein may beincluded in a kit designed for administering the biologically activemolecule to a subject. In particular implementations, the kit may bedesigned for administration of a botulinum toxin to a subject. The kitincludes a botulinum toxin present in an effective amount fortransdermal delivery thereof, and an implementation of carrier moleculelike those disclosed herein that has at least one lipophilic domainand/or hydrophobic domain and at least one CPP where the associationbetween the carrier and the botulinum toxin is non-covalent. Kitimplementations include a device for delivering the botulinum toxin tothe skin and a composition containing a carrier having at least onelipophilic domain and/or hydrophobic domain and at least one CPP. Theparticular carrier molecule and CPP(s) included in the composition maybe any disclosed in this document.

Particular kits may include a kit component designed for preparing orformulating the composition that includes the carrier and the botulinumtoxin, as well as such additional items that are needed to produce ausable formulation, or a premix that may in turn be used to produce sucha formulation. In implementations, the kit contains a pre-formulatedcomposition containing the carrier molecule and botulinum toxin; inother implementations, the kit contains a separately formulatedbotulinum toxin composition and a separately formulated carrier moleculecomposition. In other kit implementations, the kit includes materialsfor separately but in conjunction administering the botulinum toxin andthe carrier molecule implementations to a subject's skin. The kit mayalso, in various implementations contain a device for administering thecarrier molecule and/or botulinum toxin formulation to the subject viathe subject's skin. In particular implementations, the device may be askin patch.

Other components of the kit may include a device for administering thecarrier molecule and biologically active molecule to the subject via thesubject's skin. In particular implementations, the device may be a skinpatch combined with one or more tubes containing the carrier moleculeand/or biologically active molecule compositions. In particular kitimplementations, the biologically active molecule may be a botulinumtoxin. In other kit implementations, the biologically active moleculemay be any disclosed in this document. Various kit implementations caninclude one or more wipes, one or more disinfectant wipes, one or moreneedles, one or more pumps, one or more sprayers, one or more tubes, andone or more applicator devices that may include, by non-limitingexample, brushes, massagers, sonicators, and any other device for dermalapplication of a liquid or solid. Kit implementations may be designedfor use by a health care professional or may be designed to allow apatient to self-administer them.

Compositions involving carrier molecules and biologically activemolecules like those disclosed herein may be administered to a subjectusing a variety of implementations of a method of administering aparticular biologically active molecule. For example, an implementationof a method of administering a botulinum toxin to a subject includestopically applying to the skin or epithelium of the subject thebotulinum toxin in conjunction with an effective amount of a carriermolecule where the carrier molecule has at least one lipophilic domainand/or hydrophobic domain and at least one CPP, and is associatednon-covalently with the botulinum toxin. The carrier molecule and atleast one CPP may be any of those disclosed in this document. Othermethod implementations include producing a biologic effect such asmuscle paralysis, softening skin, increasing luminosity of skin,tightening skin appearance, reducing hypersecretion or sweating,altering skin pigmentation, treating neurologic pain or migraineheadache, reducing muscle spasms, preventing or reducing acne, orreducing or enhancing an immune response, by topically applying aneffective amount of a composition containing a carrier molecule andbiologically active molecule like those disclosed herein, preferably tothe skin, of a subject or patient in need of such treatment. Inaddition, the method may include producing an aesthetic or cosmeticeffect, for example, by topical application of botulinum toxin to theface instead of by conventional injection into facial muscles.

Example 1

An example of a topical preparation containing a carrier molecule andCPP along with a biologically active molecule was prepared and tested todetermine its effect. Table 1 outlines the experimental approach andresults.

TABLE 1 Control (no carrier Peptidyl Palmitoyl Component molecule)Carrier Carrier Large alkaline alkaline alkaline Molecular phosphatasephosphatase phosphatase Weight conjugate conjugate conjugate SmallMolecular salicylate salicylate salicylate Weight Carrier Molecule noneSK-2 (peptidyl- SK-1 (palmitoyl- peptide) peptide) Base CETAPHIL ®CETAPHIL ® CETAPHIL ® moisturizer moisturizer moisturizer

The alkaline phosphatase conjugate is an antibody weighting 150kilodaltons. Additional saline only controls and saline in CETAPHIL®moisturizer base manufactured by Galderma Laboratories, LP. of FortWorth, Tex. were prepared and tested as part of the experiment. TheCETAPHIL® moisturizer base contained water, glycerin, petrolatum,dicaprylyl ether, dimethicone, glyceryl stearate, cetyl acohol, prunusamygdalus dulcis (sweet almond) oil, PEG-30 stearate, tocopherylacetate, acrylates/C10-30 alkyl acrylate crosspolymer, dimethiconol,benzyl alcohol, phenoxyethanol, methylparaben, propylparaben, glycerylacrylate/acrylic acid copolymer, propylene glycol, disodium EDTA, andsodium hydroxide. The active and carrier molecules were added to thebase via micelles. The SK-1 carrier molecule had the protein sequence ofFFFILVFGGGKKRPKPG (SEQ ID NO: 27) and the SK-2 carrier molecule had theprotein sequence of palmitoyl-GGRKKRRQRRR (palmitoyl-TAT, SEQ ID NO:28).

The experiment was conducted by selecting viable porcine skin grafts ofthickness 0.045-0.055 inches that were freshly harvested, never frozen,and employed in replicates of n=5. Personnel were blinded to theidentity of formulations to be applied to the surface of the skin.Receptor fluid applied to the skin grafts (0.9% NaCl) was collected for14-16 hours after single time application of the formulation to eachtested skin graft. Receptor fluid was pipetted to a 96 well plate in 200microliter aliquots and tested in a spectrophotometer manufactured byMolecular Devices of Sunnyvale, Calif. Detection of salicylate was basedon optical density (OD) reading and was conducted first. Afterward, analiquot of 20 microliters per well of one step nitro-blue tetrazoliumand 5-bromo-4-chloro-3′-indolyphosphate (NBT-BCIP) substrate was addedto each well to visualize alkaline phosphatase activity. Timed serialmeasurements of the receptor fluid were employed to allow kineticdetermination to confirm calculated concentration from standard curves(serial 1:3 dilutions from stock 150 kilodalton antibody-alkalinephosphatase conjugate solution).

The results of the experiment are summarized in Table 2:

TABLE 2 SK-1 SK-2 No Carrier Active Active Control 2.729166672.660541667 0.187792 p < 0.01 by t-test p < 0.01 by t-test

When compared with standards, these absorbance numbers correspond to acalculated 1.8% transcutaneous flux of the biologically active moleculesand carrier molecules in the micelle base using the SK-1 (palmitoyl-TATCPP). The flux results of the two hydrophobic domain carrier moleculeswere not statistically different from one another, even across thewidely different molecular weight biologically active molecules used forthe testing. The experiment was replicated a second time and the resultswere similarly statistically significant enhanced flux of thebiologically active molecules using the carrier molecules. Thatstatistically significant flux enhancement for both large and smallmolecular weight compounds would be observed using the carrier moleculeswas an unexpected result, and indicates the effectiveness of using of acarrier molecule with a hydrophobic domain and CPP.

In places where the description above refers to particularimplementations of carrier molecule compositions, kits, and relatedmethod implementations and implementing components, sub-components,methods and sub-methods, it should be readily apparent that a number ofmodifications may be made without departing from the spirit thereof andthat these implementations, implementing components, sub-components,methods and sub-methods may be applied to other carrier moleculecompositions, kits, and related method implementations.

What is claimed is:
 1. A composition comprising: a carrier moleculecomprising at least one cell penetrating peptide (CPP), the carriermolecule comprising at least one hydrophobic domain; wherein the carrieris non-covalently associated with a biologically active molecule in oneof a micelle and a liposome.
 2. The composition of claim 1, wherein thecarrier molecule is amphiphilic.
 3. The composition of claim 1, whereinthe carrier molecule comprises at least one carbohydrate moiety.
 4. Thecomposition of claim 1, wherein the carrier molecule comprises at leastone alkyl chain.
 5. The composition of claim 1, wherein the carriermolecule comprises at least three hydrophobic amino acids.
 6. Thecomposition of claim 1, wherein the carrier molecule comprises at leastone phenylalanine.
 7. The composition of claim 1, wherein the carriermolecule comprises one of palmitoyl-gly_(p)-KKRPKPG (SEQ ID NO: 5),octanoyl-gly_(p)-KKRPKPG (SEQ ID NO: 6), oleyl-gly_(p)-KKRPKPG (SEQ IDNO: 7) and any combination thereof, where p is an integer from 0 to 20.8. The composition of claim 1, wherein the carrier molecule is selectedfrom the group consisting of FFFILVF-gly_(p)-KKRPKPG (SEQ ID NO: 1),FLVFFF-gly_(p)-KKRPKPG (SEQ ID NO: 2), KKRPKPG-gly_(p)-FLVFFF (SEQ IDNO: 3), and any combination thereof, where p is an integer from 0 to 10.9. The composition of claim 1, wherein the at least one CPP is selectedfrom the group consisting of: an HIV-TAT fragment selected from thegroup consisting of a fragment that has the formula(gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 8), a fragment that has theformula (gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 9), a fragment thathas the formula (gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 10), whereinthe subscripts p and q are each independently an integer from 0 to 20;KKRPKPG (SEQ ID NO: 17); AAVLLPVLLAAP (SEQ ID NO: 15), and anycombination thereof.
 10. The composition of claim 1, wherein thebiologically active molecule is selected from the group consisting ofVGVAPG (SEQ ID NO: 26); palmitoyl-TTS; retinyl retinoate; retinoic acid;steroid and steroidal compounds; hydroquinone; hyalonuric acid;non-steroidal anti-inflammatory drugs (NSAIDs) including naproxen,ibuprofen, and acetaminophen; skin-tightening peptides; lightactivatable moieties and compounds; ultraviolet (UV) light absorbing,blocking, or reflecting compounds; vitamins; cholesterol; drugs whichblock, influence, or interfere with neurotransmitter (such asacetylcholine) function; and any combination thereof.
 11. Thecomposition of claim 1, wherein the biologically active molecule isselected from the group consisting of: a botulinum toxin serotypeselected from the group consisting of A, B, C, D, E, F, and G;recombinant botulinum toxin; a modified botulinum toxin; a fragment of abotulinum toxin; and any combination thereof.
 12. A compositioncomprising: a biologically active molecule; and a carrier moleculecomprising at least one lipophilic domain and at least one cellpenetrating peptide (CPP), the carrier molecule further comprising oneof: at least one carbohydrate moiety; at least one alkyl chain; at leastthree hydrophobic amino acids; and any combination thereof; and whereinthe carrier molecule and biologically active molecule associatenon-covalently.
 13. The composition of claim 12, wherein the carriermolecule comprises one of palmitoyl-gly_(p)-KKRPKPG (SEQ ID NO: 5),octanoyl-gly_(p)-KKRPKPG (SEQ ID NO: 6), oleyl-gly_(p)-KKRPKPG (SEQ IDNO: 7) and any combination thereof, where p is an integer from 0 to 20.14. The composition of claim 12, wherein the carrier molecule isselected from the group consisting of FFFILVF-gly_(p)-KKRPKPG (SEQ IDNO: 1), FLVFFF-gly_(p)-KKRPKPG (SEQ ID NO: 2), KKRPKPG-gly_(p)-FLVFFF(SEQ ID NO: 3), and any combination thereof, where p is an integer from0 to
 10. 15. The composition of claim 12, wherein the at least one CPPis selected from the group consisting of: an HIV-TAT fragment selectedfrom the group consisting of a fragment that has the formula(gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 8), a fragment that has theformula (gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 9), a fragment thathas the formula (gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 10), whereinthe subscripts p and q are each independently an integer from 0 to 20;KKRPKPG (SEQ ID NO: 17); AAVLLPVLLAAP (SEQ ID NO: 15), and anycombination thereof.
 16. The composition of claim 12, wherein thebiologically active molecule is selected from the group consisting ofVGVAPG (SEQ ID NO: 26); palmitoyl-TTS; retinyl retinoate; retinoic acid;steroid and steroidal compounds; hydroquinone; hyalonuric acid;non-steroidal anti-inflammatory drugs (NSAIDs) including naproxen,ibuprofen, and acetaminophen; skin-tightening peptides; lightactivatable moieties and compounds; ultraviolet (UV) light absorbing,blocking, or reflecting compounds; vitamins; cholesterol; drugs whichblock, influence, or interfere with neurotransmitter (such asacetylcholine) function; and any combination thereof.
 17. Thecomposition of claim 12, wherein the biologically active molecule isselected from the group consisting of: a botulinum toxin serotypeselected from the group consisting of A, B, C, D, E, F, and G;recombinant botulinum toxin; a modified botulinum toxin; a fragment of abotulinum toxin; and any combination thereof.
 18. A kit foradministration of a botulinum toxin to a patient, the kit comprising: abotulinum toxin; an effective amount for transdermal delivery thereof ofa carrier molecule comprising at least one cell penetrating peptide(CPP), the carrier molecule comprising at least one hydrophobic domain;a pH buffer system adapted to maintain pH between 4.0 to 8.3; and adevice comprising the botulinum toxin, the carrier molecule, and the pHbuffer system, the device adapted to administer the botulinum toxin to apatient via the patient's skin; wherein the carrier molecule isnon-covalently associated with the botulinum toxin in one of a micelleand a liposome; and wherein one of the device, the carrier molecule, thepH buffer system, and any combination thereof are adapted to provide acontrolled release of the botulinum toxin.
 19. The kit of claim 18,wherein the device is a skin patch.
 20. The kit of claim 18, wherein thebotulinum toxin, the carrier molecule, and the pH buffer system arecomprised in a liquid, gel, cream, lotion, and ointment coupled with thedevice.